93 MET ECD shedding and poor DFS To examine whether MET degradation can be observed using the tissue microarray, it was necessary to examine overall N-terminal MET immunoreactivity alongside overall C-terminal MET immunoreactivity. Therefore, central and peripheral scores for both C- and N-terminal MET were averaged. This was valid, since the results of paired t-tests show that there are no significant differences between the center and periphery for both A2H2-3 and D1C2 (results not shown). To examine whether differences between C- and N-terminal MET immunoreactivities are associated with survival, all cancers—scored for D1C2 and A2H2-3—were assigned to one of three categories: MET negative, MET decoy receptor, or transmembranous MET with or without the ectodomain (Table 1). Negative for MET was assigned when immunoreactivity was considered to be negative for both C- and N-terminal MET. Negative immunoreactivity—for both termini—was defined using the same boundaries as previously described (18) for the C-terminus being <10% immunoreactivity in both the center and periphery. MET decoy receptor was assigned if—on average—more immunoreactivity for N-terminal than C-terminal MET was observed. Transmembranous C-terminal MET having or not having the ectodomain was assigned if—on average—more or equal amounts of C-terminal MET compared with N-terminal MET was observed. Within this category the average % of cells subjective to ectodomain shedding was calculated by subtracting the average % of A2H2-3 positive cells from the average % of D1C2 positive cells (Table 1). Association between MET ectodomain shedding and survival The optimal threshold for ectodomain shedding in view of survival analysis was determined using the receiver operating characteristic curve analysis for disease-free survival and overall survival. Only cancers positive for transmembranous C-terminal MET were included and the area under the curve was used as a performance measure. Survival analyses Overall survival was defined as the time in months from the date of primary surgery to: the date of death due to any cause or the cutoff time (set at 60 months). Disease-free survival was defined as the time in months from the date of primary surgery to: the date of first evidence of any disease (local, regional, distant, or secondary 4
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